Polynucleotides that encode the calcitonin gene-related peptide receptor coponent factor (HOUNDC44)

ABSTRACT

Human CGRP-RCF polypeptides and DNA (RNA) encoding such CGRP-RCF and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such CGRP-RCF for the treatment of diabetes, migrane, pain and inflammation, Parkinson&#39;s disease, acute heart failure, hypotension, urinary retention, osteoporosis, hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychosis, depression, vomiting, benign prostatic hypertrophy, Paget&#39;s disease, obesity, cancer, gigantism and the like. Antagonists against such CGRP-RCF and their use as a therapeutic to treat diabetes, migrane, pain and inflammation, Parkinson&#39;s disease, acute heart failure, hypotension, urinary retention, osteoporosis, hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychosis, depression, vomiting, benign prostatic hypertrophy, Paget&#39;s disease, obesity, cancer, gigantism and the like are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the CGRP-RCF and for detecting altered levels of the polypeptide in a host.

FIELD OF THE INVENTION

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; processes for making the polynucleotides and thepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of the polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of human Calcitonin Gene-relatedPeptide Receptor Component Factor, hereinafter referred to as"CGRP-RCF".

BACKGROUND OF THE INVENTION

Calcitonin gene related peptide (CGRP) is a 37 amino acidcarboxyl-amidated neuropeptide secreted by the nerves of the central andperipheral nervous systems and exists as highly homologous α or βisoforms in both human and rat (Amara, et al. Nature 298:240-244 (1982);Amara, et al. Science 229:1094-1097 (1985)). α-and β-CGRP display verysimilar biological activities, including peripheral and cerebralvasodilation (Brain, et al., Nature 313:54-56 (1985)), cardiacacceleration, (Sigrist, et al., Endocrinology 119:381-389 (1986)),regulation of calcium metabolism (Grunditz, et al., Endocrinology119:2313-2324 (1986)), reduction of intestinal motility (Fargeas, etal., Peptides 6:1167-1171(1985)), regulation of glucose metabolism(reduction of insulin secretion and insulin sensitivity) (Hermansen, etal., Reg. Peptides 27:149-157) (1990)), diminution of appetite (Molina,et al., Diabetes 39:260-265 (1990)) and reduction of growth hormonerelease (Tannenbaum, et al., Endocrinology 116:2685-2687 (1985)). Thetwo CGRP peptides differ by three amino acids in humans and one aminoacid in rats. The amino acid sequences of CGRP peptides are wellconserved among species and can be considered as members of a family ofpeptides including the related peptides amylin (46% homology), salmoncalcitonin (32% homology), and adrenomedullin (24% homology). Thesepeptides in general have N-terminal ring structures of 6-7 amino acidsinvolving a disulfide bridge and an amidated C-terminal end (Muff, etal., Eur J Endocrinol. 133:17-20 (1995); Goodman, et al., Life Sci.38:2169-2178 (1986); Poyner, D. R. Pharmac. Ther. 56:23-51 (1992)).

CGRP peptides are localized predominantly in sensory afferent nerves andcentral neurons (Goodman, et al., Life Sci. 38:2169-2178 (1986); Poyner,D. R. Pharmac. Ther. 56:23-51 (1992)). When released from the cell, thepeptides initiate their biological responses by binding to specific cellsurface receptors which are predominantly coupled to the activation ofadenylyl cyclase (Brain, et al., Nature 313:54-56 (1985); Kubota, et al,Biochem Biophys Res Commun 132:88-94 (1985)). CGRP receptors have beenidentified and pharmacologically evaluated in several tissues and cells,including brain, cardiovascular, endothelial and smooth muscle (Poyner,D. R. Pharmac. Ther. 56:23-51 (1992)). Multiple CGRP receptors have beenobserved, based on pharmacological properties. These receptors they aredivided into at least two subtypes and denoted as CGRP₁ and CGRP₂,according to the classification of Dennis and colleagues (Dennis, etal., Brain Res. 539:59-66 (1991)). CGRP (Molina, et al. Diabetes39:260-265 (1990); Tannenbaum, et al., Endocrinology 116:2685-2687(1985); Muff, et al., Eur J Endocrinol. 133: 17-20( 1995); Goodman, etal., Life Sci. 38:2 169-2178 (1986); Poyner, D. R., Pharmac. Ther.56:23-51 (1992); Kubota, et al., Biochem Biophys Res Commun 132:88-94(1985); Dennis, et al., Brain Res. 539:59-66 (1991); Adams, et al.,Science 252:1651-1656 (1991); Adams, et al., Nature 355:632-634 (1992);Adams, et al., Nature 377:3-174 (1995); Chang, et al., Neuron11:1187-1195 (1993); Fluhmann, et al., Biochem. Biophy. Res. Comm.206:341-347 (1995); Jelinek, et al., Science 259:1614-1616 (1993);Kozak, M., Proc. Natl. Acad. Sci. (USA), 92:2662-2666 (1995); Aiyar, etal., Mol. Cell. Bio. 131:75-86 (1994); Sambrook, et al., MolecularCloning. Cold Spring Harbor Laboratory Press (1989); Nuovo, J. G., PCRin situ Hybridization: protocols and applications. Raven Press, New York(1992); Aiyar, et al. Endocrinology 129:965-969 (1991); Lin, et al.,Science 254:1022-1024 (1991); Hirata, et al., Biochem. Biophy. Res.Commun. 151:1113-1121 (1988); Nawa, et al., Nature 312:729-734 (1984);Kozak, M. J., Mol. Biol. 196:947-950 (1987); Dohlman, et al., Annu. Rev.Biochem. 60:653-688 (1991); Jackson, T., Pharmocol. Ther. 50:425-442(1991); Ishihara, et al., EMBO J., 10:1635-1641 (1991); Juppner, et al.,Science 254:1024-1026 (1991); Thorens, B., Proc. Natl. Acad. Sci.U.S.A., 89:8641-8645 (1992); van Valen, et al., Neurosci. Lett.119:195-198 (1990); Stangl, et al., Endocrinology 132:744-750 (1993);Kitamura, et al., FEBS Lett., 338:306-310 (1994)), which lacks 7N-terminal amino acid residues, is a selective antagonist of CGRP₁receptors, whereas the linear analog of CGRP, diacetoamidomethylcysteine CGRP ({Cys(ACM)2,7}CGRP), is a selective agonist of CGRP₂receptors (Dennis, et al., Brain Res. 539:59-66 (1991)).

As indicated above, CGRP has a plethora of functions in the body and isknown to be the most potent vasodilator and neuromodulator. Although werecently reported the cDNA encoding the human CGRP-type I receptor(Aiyar et al., Journal of Biological Chemistry 271:11325-11329 (1996)),we observed during our characterization studies that the receptor didnot confer CGRP responsiveness in Xenopus oocytes, in transientlyexpressing COS cells, and in stably expressing Baculovirus andDrosophila cells. In contrast to these observations, we are able toconfer reasonable levels of CGRP responsiveness in stably transfectedhuman embyonic kidney 293 cells. These observations suggest therequirement of an additional human complementary factor for functionalcoupling of the CGRP receptor in all of these heterologous systems.

Recently, Luebke et al., (Proc. Natl. Acad. Science, 93:3455-3460(1996)) used an expression cloning strategy to isolate a guinea pig cDNAthat encodes a protein (CGRP-receptor component protein) that confersCGRP responsiveness in oocytes. Utilizing this information and using theexpressed sequence tag (EST) analysis (Adams, et al., Science252:1651-1656 (1991); Adams, et al., Nature 355:632-634 (1992); Adams,et al., Nature 377:3-174 (1995)) we identified the human homologue ofthe guinea pig CGRP-RCP. A full length cDNA clone is identified from ahuman adipocytes osteoclastoma cDNA library.

Although numerous groups have indicated the absolute requirement forcomplementary RCFs for functional signaling in heterologous systems forseveral 7TM receptors (JBC 266:12560, 1991 and FEBS Lett. 291:208,1991), none were able to successfully isolate the cDNA encoding thesespecific factors.

The identification and isolation of a complementary human factorneccessary for expression of the CGRP type I receptor in heterologoussystems, provides an opportunity to delineate more exactly or enhancethe signaling pathways that are activated by the receptor. This alsooffers a novel approach to identify functionally the increased number oforphan cDNAs encoding suspected G-protein linked receptors whose ligandsare still unknown.

Clearly, there is a need for factors necessary for correct signaltransduction of CGRP receptors (or even other G-protein coupledreceptors) and their roles in dysfunction and disease. There is a need,therefore, for identification and characterization of such factors whichcan play a role in preventing, ameliorating or correcting dysfunctionsor diseases, such as, but not limited to, diabetes, migrane, pain andinflammation.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelCGRP-RCF by homology between the amino acid sequence set out in FIG. 1and known amino acid sequences of other proteins such as guinea pigCGRP-RCP described in by Luebke in the Proc. Natl. Acad. Sci.93:3455-3460 (1996).

It is a further object of the invention, moreover, to providepolynucleotides that encode CGRP-RCF, particularly polynucleotides thatencode the polypeptide herein designated CGRP-RCF.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding human CGRP-RCF in thesequence set out in FIG. 1.

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA contained in ATCC Deposit No. 98105.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding human CGRP-RCF, includingmRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect ofthe invention, biologically, diagnostically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of human CGRP-RCF.

It also is an object of the invention to provide CGRP-RCF polypeptides,particularly human CGRP-RCF polypeptides, that may be employed fortherapeutic purposes, for example, to treat diabetes, migrane, pain andinflammation, Parkinson's disease, acute heart failure, hypotension,urinary retention, osteoporosis, hypertension, angina pectoris,myocardial infarction, ulcers, asthma, allergies, psychosis, depression,vomiting, benign prostatic hypertrophy, Paget's disease, obesity,cancer, gigantism and the like.

In accordance with this aspect of the invention there are provided novelpolypeptides of human origin referred to herein as CGRP-RCF as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of human CGRP-RCF encoded by naturally occurringalleles of the human CGRP-RCF gene.

In accordance with another aspect of the present invention there areprovided methods of screening for compounds which bind to and activateor inhibit activation of the CGRP receptor or CGRP/CGRP-RCF receptorpolypeptides and for receptor ligands.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned CGRP-RCF polypeptidescomprising culturing host cells having expressibly incorporated thereinan exogenously-derived human CGRP-RCF-encoding polynucleotide underconditions for expression of human CGRP-RCF in the host and thenrecovering the expressed polypeptide.

In accordance with another object the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for, among other things: assessing CGRP-RCF expression in cells bydetermining CGRP-RCF polypeptides or CGRP-RCF-encoding mRNA; treatingdiabetes, migrane, pain and inflammation, Parkinson's disease, acuteheart failure, hypotension, urinary retention, osteoporosis,hypertension, angina pectoris, myocardial infarction, ulcers, asthma,allergies, psychosis, depression, vomiting, benign prostatichypertrophy, Paget's disease, obesity, cancer, gigantism and the like,in vitro, ex vivo or in vivo by exposing cells to CGRP-RCF polypeptidesor polynucleotides as disclosed herein; assaying genetic variation andaberrations, such as defects, in CGRP-RCF genes; and administering aCGRP-RCF polypeptide or polynucleotide to an organism to augmentCGRP-RCF function or remediate CGRP-RCF dysfunction.

In accordance with still another embodiment of the present inventionthere is provided a process of using such activating compounds tostimulate the CGRP receptor polypeptide for the treatment of conditionsrelated to the under-expression of the CGRP-RCF.

In accordance with another aspect of the present invention there isprovided a process of using such inhibiting compounds for treatingconditions associated with over-expression of the CGRP-RCF.

In accordance with yet another aspect of the present invention there isprovided non-naturally occurring synthetic, isolated and/or recombinantCGRP-RCF polypeptides which are fragments, consensus fragments and/orsequences having conservative amino acid substitutions, of at least onedomain of the CGRP-RCF of the present invention, such that the CGRP-RCFmay bind to CGRP receptor ligands or to CGRP receptor, or which may alsomodulate, quantitatively or qualitatively, CGRP receptor ligand binding.

In accordance with still another aspect of the present invention thereare provided synthetic or recombinant CGRP-RCF polypeptides,conservative substitution and derivatives thereof, antibodies,anti-idiotype antibodies, compositions and methods that can be useful aspotential modulators of CGRP-RCF function, by binding to CGRP receptorligands or CGRP receptor or modulating ligand binding, which may be usedin diagnostic, therapeutic and/or research applications.

It is still another object of the present invention to providesynthetic, isolated or recombinant polypeptides which are designed toinhibit or mimic various CGRP-RCF or fragments thereof.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided probes that hybridize tohuman CGRP-RCF sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against CGRP-RCF polypeptides.In certain particularly preferred embodiments in this regard, theantibodies are highly selective for human CGRP-RCF.

In accordance with another aspect of the present invention, there areprovided CGRP-RCF and CGRP-RCF/CGRP receptor agonists. Among preferredagonists are molecules that mimic CGRP-RCF, that bind to CGRP receptormolecules or CGRP-RCF, and that elicit or augment CGRP-RCF-inducedresponses. Also among preferred agonists are molecules that interactwith CGRP-RCF or CGRP-RCF/CGRP-RCF receptor, or with other modulators ofCGRP-RCF activities, and thereby potentiate or augment an effect ofCGRP-RCF or more than one effect of CGRP-RCF.

In accordance with yet another aspect of the present invention, thereare provided CGRP-RCF and CGRP-RCF/CGRP receptor antagonists. Amongpreferred antagonists are those which mimic CGRP-RCF so as to bind toCGRP receptor or CGRP-RCF but inhibit CGRP-RCF-induced response or morethan one CGRP-RCF-induced response. Also among preferred antagonists aremolecules that bind to or interact with CGRP-RCF or CGRP/CGRP-RCFreceptor so as to inhibit an effect of CGRP-RCF or more than one effectof CGRP-RCF or which prevent expression of CGRP-RCF.

In a further aspect of the invention there are provided compositionscomprising a CGRP-RCF polynucleotide or a CGRP-RCF polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprise aCGRP-RCF polynucleotide for expression of a CGRP-RCF polypeptide in ahost organism for treatment of disease. Particularly preferred in thisregard is expression in a human patient for treatment of a dysfunctionassociated with aberrant endogenous activity of CGRP-RCF.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawing depicts certain embodiments of the invention. Itis illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the nucleotide and deduced amino acid sequence of humanCGRP-RCF.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe examples. The explanations are provided as a convenience and are notlimitative of the invention.

DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

For analytical purposes, typically, 1 μg of plasmid or DNA fragment isdigested with about 2 units of enzyme in about 20 μl of reaction buffer.For the purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzymein proportionately larger volumes.

Appropriate buffers and substrate amounts for particular restrictionenzymes are described in standard laboratory manuals, such as thosereferenced below, and they are specified by commercial suppliers.

Incubation times of about 1 hour at 37° C. are ordinarily used, butconditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a region that regulates transcription ortranslation or other processes important to expression of thepolypeptide in a host cell, or a polynucleotide comprising both a regionthat encodes a polypeptide and a region operably linked thereto thatregulates expression.

Genetic elements may be comprised within a vector that replicates as anepisomal element; that is, as a molecule physically independent of thehost cell genome. They may be comprised within mini-chromosomes, such asthose that arise during amplification of transfected DNA by methotrexateselection in eukaryotic cells. Genetic elements also may be comprisedwithin a host cell genome; not in their natural state but, rather,following manipulation such as isolation, cloning and introduction intoa host cell in the form of purified DNA or in a vector, among others.

ISOLATED means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both.

For example, a naturally occurring polynucleotide or a polypeptidenaturally present in a living animal in its natural state is not"isolated," but the same polynucleotide or polypeptide separated fromthe coexisting materials of its natural state is "isolated", as the termis employed herein. For example, with respect to polynucleotides, theterm isolated means that it is separated from the chromosome and cell inwhich it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

LIGATION refers to the process of forming phosphodiester bonds betweentwo or more polynucleotides, which most often are double stranded DNAs.Techniques for ligation are well known to the art and protocols forligation are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Maniatis et al., pg. 146, as cited below.

OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often theterm refers to single-stranded deoxyribonucleotides, but it can refer aswell to single-or double-stranded ribonucleotides, RNA:DNA hybrids anddouble-stranded DNAs, among others.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides,often are synthesized by chemical methods, such as those implemented onautomated oligonucleotide synthesizers. However, oligonucleotides can bemade by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells andorganisms.

Initially, chemically synthesized DNAs typically are obtained without a5' phosphate. The 5' ends of such oligonucleotides are not substratesfor phosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP.

The 3' end of a chemically synthesized oligonucleotide generally has afree hydroxyl group and, in the presence of a ligase, such as T4 DNAligase, readily will form a phosphodiester bond with a 5' phosphate ofanother polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5' phosphates of the other polynucleotide(s) prior toligation.

PLASMIDS generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Starting plasmids disclosed herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions.

In addition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are"polynucleotides" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses, such as processing and other posttranslational modifications,but also by chemical modification techniques which are well known to theart. Even the common modifications that occur naturally in polypeptidesare too numerous to list exhaustively here, but they are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature, and they are well known to those ofskill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663:48-62 (1992).

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcell often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

(1) A polynucleotide that differs in nucleotide sequence from another,reference polynucleotide. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical.

As noted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

(2) A polypeptide that differs in amino acid sequence from another,reference polypeptide. Generally, differences are limited so that thesequences of the reference and the variant are closely similar overalland, in many region, identical.

A variant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

FUSION PROTEINS: EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in fusion protein is thoroghlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,shIL5-α has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,Bennett et al., Journal of Molecular Recognition, 8:52-58 (1995) andJohanson et al., The Journal of Biological Chemistry, 270, No. 16,9459-9471 (1995).

Thus, this invention also relates to genetically engineered solublefusion proteins comprised from CGRP-RCF, or a portion thereof, and ofvarious portions of the constant regions of heavy or light chains ofimmunoglobulins of various subclass (IgG, IgM, IgA, IgE). Preferred asimmunoglobulin is the constant part of the heavy chain of human IgG,particularly IgG 1, where fusion takes place at the hinge region. In aparticular embodiment, the Fc part can be removed in a simple way by acleavage sequence which is also incorporated and can be cleaved withfactor Xa. Furthermore, this invention relates to processes for thepreparation of these fusion by genetic engineering, and to the usethereof for diagnosis and therapy. An yet further aspect of theinvention also relates to polynucleotide encoding such fusion proteins.

BINDING MOLECULES (or otherwise called INTERACTION MOLECULES or RECEPTORCOMPONENT FACTORS) refer to molecules other than CGRP receptor ligandsor CGRP receptor that specifically bind to or interact with polypeptidesof the present invention. Such binding molecules are a part of thepresent invention. BINDING MOLECULES also may be non-naturallyoccurring, such as antibodies and antibody-derived reagents that bindspecifically to polypeptides of the invention.

CGRP-RCF/CGRP RECEPTOR SYSTEM or CGRP-RCF/CGRP RECEPTOR or simplyRECEPTOR SYSTEM as used herein refers to a complex formed between CGRPreceptor and CGRP-RCF.

DESCRIPTION OF THE INVENTION

The present invention relates to novel CGRP-RCF polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel human CGRP-RCF, which is related by aminoacid sequence homology to guinea pig CGRP-RCP polypeptide. The inventionrelates especially to CGRP-RCF having the nucleotide and amino acidsequences set out in FIG. 1, and to the CGRP-RCF nucleotide and aminoacid sequences of the human cDNA in ATCC Deposit No. 98105, which isherein referred to as "the deposited clone" or as the "cDNA of thedeposited clone." It will be appreciated that the nucleotide and aminoacid sequences set out in FIG. 1 are obtained by sequencing the cDNA ofthe deposited clone. Hence, the sequence of the deposited clone iscontrolling as to any discrepancies between the two and any reference tothe sequences of FIG. 1 include reference to the sequence of the humancDNA of the deposited clone.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the CGRP-RCF polypeptidehaving the deduced amino acid sequence of FIG. 1.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1, a polynucleotide of the present inventionencoding human CGRP-RCF polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA from human adipocytes from osteoclastoma as starting material.Illustrative of the invention, the polynucleotide set out in FIG. 1 isdiscovered in a cDNA library derived from human adipocytes fromosteoclastoma using the expressed sequence tag (EST) analysis (Adams, etal., Science 252:1651-1656 (1991); Adams, et al., Nature 355:632-634(1992); Adams, et al., Nature 377 Supp, 3-174) (1995)).

Human CGRP-RCF of the invention is structurally related to the guineapig CGRP-RCP, as shown by the results of sequencing the cDNA encodinghuman CGRP-RCF in the deposited clone. The cDNA sequence thus obtainedis set out in FIG. 1. SEQ ID NO: 1. It contains an open reading frameencoding a protein of 148 amino acid residues with a deduced molecularweight of about 17.7 kDa. CGRP-RCF of FIG. 1 has about 88% identity andabout 94% similarity with the amino acid sequence of guinea pigCGRP-RCP.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1. SEQ ID NO: 1.It also may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of the DNA of FIG. 1.

Polynucleotides of the present invention which encode the polypeptide ofFIG. 1 may include, but are not limited to the coding sequence for themature polypeptide, by itself; the coding sequence for the maturepolypeptide and additional coding sequences, such as those encoding aleader or secretory sequence, such as a pre-, or pro- or prepro-proteinsequence; the coding sequence of the mature polypeptide, with or withoutthe aforementioned additional coding sequences, together withadditional, non-coding sequences, including for example, but not limitedto introns and non-coding 5' and 3' sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing--including splicing and polyadenylation signals, forexample--ribosome binding and stability of mRNA; additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, for instance, the polypeptidemay be fused to a marker sequence, such as a peptide, which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker sequence is a hexa-histidinepeptide, such as the tag provided in the pQE vector (Qiagen, Inc.),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci., USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. The HA tag corresponds to an epitope derived ofinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984), for instance.

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlythe human CGRP-RCF having the amino acid sequence set out in FIG. 1. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,interrupted by introns) together with additional regions, that also maycontain coding and/or non-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1. A variant of the polynucleotide may be a naturally occurringvariant such as a naturally occurring allelic variant, or it may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the polynucleotide may be made by mutagenesistechniques, including those applied to polynucleotides, cells ororganisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of CGRP-RCF set out in FIG. 1; variants, analogs, derivativesand fragments thereof, and fragments of the variants, analogs andderivatives.

Further particularly preferred in this regard are polynucleotidesencoding CGRP-RCF variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the CGRP-RCF polypeptide of FIG. 1 in which several, afew, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, which donot alter the properties and activities of the CGRP-RCF. Also especiallypreferred in this regard are conservative substitutions. Most highlypreferred are polynucleotides encoding polypeptides having the aminoacid sequence of FIG. 1, without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotide encoding the CGRP-RCFpolypeptide having the amino acid sequence set out in FIG. 1, andpolynucleotides which are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical to a polynucleotide encoding theCGRP-RCF polypeptide of the human cDNA of the deposited clone andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical to the same are particularly preferred, and amongthese particularly preferred polynucleotides, those with at least 95%are especially preferred. Furthermore, those with at least 97% arehighly preferred among those with at least 95%, and among these thosewith at least 98% and at least 99% are particularly highly preferred,with at least 99% being the more preferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the cDNA of FIG. 1.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding CGRP-RCF and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the human CGRP-RCF gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

For example, the coding region of the CGRP-RCF gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofhuman cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease, as further discussed herein relatingto polynucleotide assays, inter alia.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may facilitateprotein trafficking, may prolong or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited materials

A deposit containing a human CGRP-RCF cDNA has been deposited with theAmerican Type Culture Collection, as noted above. Also as noted above,the human cDNA deposit is referred to herein as "the deposited clone" oras "the cDNA of the deposited clone."

The deposited clone is deposited with the American Type CultureCollection, 12301 Park Lawn Drive, Rockville, Md. 20852, U.S.A., on Jul.17, 1996, and assigned ATCC Deposit No. 98105 (Escherichia colipHOUDC44/SolR).

The deposited material is a pBluescript SK (-) plasmid (Stratagene, LaJolla, Calif.)* that contains the full length CGRP-RCF cDNA, referred toas pHOUDC44 upon deposit.

The deposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a human CGRP-RCF polypeptidewhich has the deduced amino acid sequence of FIG. 1. SEQ ID NO:2.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms "fragment," "derivative" and "analog" whenreferring to the polypeptide of FIG. 1, means a polypeptide whichretains essentially the same biological function or activity as suchpolypeptide, i.e. functions as a CGRP-RCF, or retains the ability tobind the CGRP receptor ligand or the CGRP receptor even though thepolypeptide does not function as a CGRP-RCF, for example, a soluble formof CGRP-RCF. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, or (ii) one in whichone or more of the amino acid residues includes a substituent group, or(iii) one in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of CGRP-RCF setout in FIG. 1, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments. Alternatively,particularly preferred embodiments of the invention in this regard arepolypeptides having the amino acid sequence of the CGRP-RCF, variants,analogs, derivatives and fragments thereof, and variants, analogs andderivatives of the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the CGRP-RCF polypeptide ofFIG. 1, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or noamino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the CGRP-RCF. Also especially preferred in this regard areconservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 1 without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 80% identity to the polypeptide of SEQ ID NO:2 andmore preferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of SEQ ID NO:2 and still more preferably atleast 95% similarity (still more preferably at least 95% identity) tothe polypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.Moreover, also known in the an is "identity" which means the degree ofsequence relatedness between two polypeptide or two polynucleotidessequences as determined by the identity of the match between two stringsof such sequences. Both identity and similarity can be readilycalculated (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, NewYork, 1991). While there exist a number of methods to measure identityand similarity between two polynucleotide or polypeptide sequences, theterms "identity" and "similarity" are well known to skilled artisans(Sequence Analysis in Molecular Biology, yon Heinje, G., Academic Press,1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988). Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to disclosed in Guide to Huge Computers, Martin J. Bishop, ed.,Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAMJ. Applied Math., 48: 1073 (1988). Preferred methods to determineidentity are designed to give the largest match between the twosequences tested. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J. Molec. Biol. 215:403 (1990)).

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of CGRP-RCF, most particularlyfragments of the CGRP-RCF having the amino acid set out in FIG. 1, andfragments of variants and derivatives of the CGRP-RCF of FIG. 1.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned CGRP-RCF polypeptides and variants or derivativesthereof.

Such fragments may be "free-standing," i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a CGRP-RCFpolypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the CGRP-RCFfragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from CGRP-RCF.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 5-15, 10-20, 15-40,30-55, 41-75, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 110-113amino acids long.

In this context about includes the particularly recited range and rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes. For instance, about 40-90 aminoacids in this context means a polypeptide fragment of 40 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 90 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 40minus several amino acids to 90 plus several amino acids to as narrow as40 plus several amino acids to 90 minus several amino acids.

Highly preferred in this regard are the recited ranges plus or minus asmany as 5 amino acids at either or at both extremes. Particularly highlypreferred are the recited ranges plus or minus as many as 3 amino acidsat either or at both the recited extremes. Especially particularlyhighly preferred are ranges plus or minus 1 amino acid at either or atboth extremes or the recited ranges with no additions or deletions. Mosthighly preferred of all in this regard are fragments from about 5-15,10-20, 15-40, 30-55, 41-75, 41-80, 41-90, 50-100, 75-100, 90-115,100-125, and 110-113 amino acids long.

Among especially preferred fragments of the invention are truncationmutants of CGRP-RCF. Truncation mutants include CGRP-RCF polypeptideshaving the amino acid sequence of FIG. 1, or of variants or derivativesthereof, except for deletion of a continuous series of residues (thatis, a continuous region, part or portion) that includes the aminoterminus, or a continuous series of residues that includes the carboxylterminus or, as in double truncation mutants, deletion of two continuousseries of residues, one including the amino terminus and one includingthe carboxyl terminus. Fragments having the size ranges set out aboutalso are preferred embodiments of truncation fragments, which areespecially preferred among fragments generally.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of CGRP-RCF.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions("alpha-regions"), beta-sheet and beta-sheet-forming regions("beta-regions"), turn and turn-forming regions ("turn-regions"), coiland coil-forming regions ("coil-regions"), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of CGRP-RCF.

Among highly preferred fragments in this regard are those that compriseregions of CGRP-RCF that combine several structural features, such asseveral of the features set out above. In this regard, the regionsdefined by the residues about 10 to about 20, about 40 to about 50,about 70 to about 90 and about 100 to about 113 of FIG. 1, which all arecharacterized by amino acid compositions highly characteristic ofturn-regions, hydrophilic regions, flexible-regions, surface-formingregions, and high antigenic index-regions, are especially highlypreferred regions. Such regions may be comprised within a largerpolypeptide or may be by themselves a preferred fragment of the presentinvention, as discussed above. It will be appreciated that the term"about" as used in this paragraph has the meaning set out aboveregarding fragments in general.

Further preferred regions are those that mediate activities of CGRP-RCF.Most highly preferred in this regard are fragments that have a chemical,biological or other activity of CGRP-RCF, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Highly preferred in this regard are fragments that containregions that are homologs in sequence, or in position, or in bothsequence and to active regions of related polypeptides, such as therelated polypeptides of guinea pig CGRP-RCP. Among particularlypreferred fragments in these regards are truncation mutants, asdiscussed above.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespondent to the preferred fragments, as discussed above.

Vectors, host cells, expression

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate, polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation also may be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. cited above, which is illustrative of the manylaboratory manuals that detail these techniques.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case viralpropagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskilled in the art, are set forth in great detail in Sambrook et al.cited elsewhere herein.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. It will beunderstood that numerous promoters not mentioned are suitable for use inthis aspect of the invention are well known and readily may be employedby those of skill in the manner illustrated by the discussion and theexamples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as repressor binding sites and enhancers, amongothers.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. These vectors are listed solelyby way of illustration of the many commercially available and well knownvectors that are available to those of skill in the art for use inaccordance with this aspect of the present invention. It will beappreciated that any other plasmid or vector suitable for, for example,introduction, maintenance, propagation or expression of a polynucleotideor polypeptide of the invention in a host may be used in this aspect ofthe invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase ("CAT") transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-8 and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous sarcoma virus ("RSV"), and metallothionein promoters, suchas the mouse metallothionein-I promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al. BASIC METHODS IN MOLECULARBIOLOGY, (1986).

Constructs in host cells can be used in a conventional manner to producethe gene product encoded by the recombinant sequence. Alternatively, thepolypeptides of the invention can be synthetically produced byconventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector. Among suitable promoters are those derived from the genesthat encode glycolytic enzymes such as 3-phosphoglycerate kinase("PGK"), a-factor, acid phosphatase, and heat shock proteins, amongothers. Selectable markers include the ampicillin resistance gene of E.coli and the trp 1 gene of S. cerevisiae.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5' to aribosome binding site. The ribosome binding site will be 5' to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiating AUG. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal and a transcription termination signalappropriately disposed at the 3' end of the transcribed region.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Suitable prokaryotic hosts for propagation, maintenance or expression ofpolynucleotides and polypeptides in accordance with the inventioninclude Escherichia coli, Bacillus subtilis and Salmonella typhimurium.Various species of Pseudomonas, Streptomyces, and Staphylococcus aresuitable hosts in this regard. Moreover, many other hosts also known tothose of skill may be employed in this regard.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 "backbone" sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the COS-7lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23:175 (1981). Other cell lines capable of expressing a compatible vectorinclude for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHKcell lines.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5' flanking non-transcribedsequences that are necessary for expression. In certain preferredembodiments in this regard DNA sequences derived from the SV40 splicesites, and the SV40 polyadenylation sites are used for requirednon-transcribed genetic elements of these types.

The CGRP-RCF polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography ("HPLC") is employed for purification. Well knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and orpurification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

CGRP-RCF polynucleotides and polypeptides may be used in accordance withthe present invention for a variety of applications, particularly thosethat make use of the chemical and biological properties of CGRP-RCF.Additional applications relate to diagnosis and to treatment ofdisorders of cells, tissues and organisms. These aspects of theinvention are illustrated further by the following discussion.

Polynucleotide assays

This invention is also related to the use of the CGRP-RCFpolynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a mutated form ofCGRP-RCF associated with a dysfunction will provide a diagnostic toolthat can add or define a diagnosis of a disease or susceptibility to adisease which results from under-expression over-expression or alteredexpression of CGRP-RCF. Individuals carrying mutations in the humanCGRP-RCF gene may be detected at the DNA level by a variety oftechniques. Nucleic acids for diagnosis may be obtained from a patient'scells, such as from blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR prior to analysis. PCR (Saiki etal., Nature, 324: 163-166 (1986)). RNA or cDNA may also be used in thesame ways. As an example, PCR primers complementary to the nucleic acidencoding CGRP-RCF can be used to identify and analyze CGRP-RCFexpression and mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled CGRP-RCF RNA or alternatively,radiolabeled CGRP-RCF antisense DNA sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms ("RFLP") and Southern blottingof genomic DNA.

In accordance with a further aspect of the invention, there is provideda process for determining or diagnosing diabetes, migrane, pain andinflammation, Parkinson's disease, acute heart failure, hypotension,urinary retention, osteoporosis, hypertension, angina pectoris,myocardial infarction, ulcers, asthma, allergies, psychosis, depression,vomiting, benign prostatic hypertrophy, Paget's disease, obesity,cancer, gigantism and the like or a susceptibility to the foregoingdiseases. Thus, a mutation in CGRP-RCF indicates a susceptibility totreat diabetes, migrane, pain and intimation, Parkinson's disease, acuteheart failure, hypotension, urinary retention, osteoporosis,hypertension, angina pectoris, myocardial infarction, ulcers, asthma,allergies, psychosis, depression, vomiting, benign prostatichypertrophy, Paget's disease, obesity, cancer, gigantism and the like,and the nucleic acid sequences described above may be employed in anassay for ascertaining such susceptibility. Thus, for example, the assaymay be employed to determine a mutation in a human CGRP-RCF protein asherein described, such as a deletion, truncation, insertion, frameshift, etc., with such mutation being indicative of a susceptibility tothe foregoing diseases.

A mutation may be ascertained for example, by a DNA sequencing assay.Tissue samples, including but not limited to blood samples are obtainedfrom a human patient. The samples are processed by methods known in theart to capture the RNA. First strand cDNA is synthesized from the RNAsamples by adding an oligonucleotide primer consisting of polythymidineresidues which hybridize to the polyadenosine stretch present on themRNA's. Reverse transcriptase and deoxynucleotides are added to allowsynthesis of the first strand cDNA. Primer sequences are synthesizedbased on the DNA sequence of the protein of the invention. The primersequence is generally comprised of at least 15 consecutive bases, andmay contain at least 30 or even 50 consecutive bases.

Thus, the detection of a specific DNA sequence and/or quantitation ofthe level of the sequence may be achieved by methods such ashybridization, RNase protection, chemical cleavage, direct DNAsequencing or the use of restriction enzymes, (e.g., RestrictionFragment Length Polymorphisms (RFLP)) and Southern blotting of genomicDNA. The invention provides a process for diagnosing diabetes, migrane,pain and inflammation, Parkinson's disease, acute heart failure,hypotension, urinary retention, osteoporosis, hypertension, anginapectoris, myocardial infarction, ulcers, asthma, allergies, psychosis,depression, vomiting, benign prostatic hypertrophy, Paget's disease,obesity, cancer, gigantism and the like, comprising determining from asample derived from a patient an abnormally decreased or increased levelof expression of polynucleotide having the sequence of FIG. 1 (SEQ IDNO: 1). Decreased or increased expression of polynucleotide can bemeasured using any on of the methods well known in the art for thequantation of polynucleotides, such as, for example, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomallocation. As an example of how this is performed, CGRP-RCF DNA isdigested and purified with QIAEX II DNA purification kit (QIAGEN, Inc.,Chatsworth, Calif.) and ligated to Super Cos1 cosmid vector (STRATAGENE,La Jolla, Calif.). DNA is purified using Qiagen Plasmid Purification Kit(QIAGEN Inc., Chatsworth, Calif.) and 1 mg is labeled by nicktranslation in the presence of Biotin-dATP using BioNick Labeling Kit(GibcoBRL, Life Technologies Inc., Gaithersburg, Md.). Biotinilation isdetected with GENETECT Detection System (CLONTECH Laboratories, Inc.Palo Alto, Calif.). In situ Hybridization is performed on slides usingONCOR Light Hybridization Kit (ONCOR, Gaithersberg, Md.) to detectsingle copy sequences on metaphase chromosomes. Peripheral blood ofnormal donors is cultured for three days in RPMI 1640 supplemented with20% FCS, 3% PHA and penicillin/streptomycin, synchronized with 10⁻⁷ Mmethotrexate for 17 hours and washed twice with unsupplemented RPMI.Cells are incubated with 10⁻³ M thymidine for 7 hours. The cells arearrested in metaphase after 20 minutes incubation with colcemid (0.5μg/ml) followed by hypotonic lysis in 75 mM KCl for 15 minutes at 37° C.Cell pellets are then spun out and fixed in Carnoy's fixative (3:1methanol/acetic acid).

Metaphase spreads are prepared by adding a drop of the suspension ontoslides and aid dried. Hybridization is performed by adding 100 ng ofprobe suspended in 10 ml of hybridization mix (50% formamide, 2×SSC, 1%dextran sulfate) with blocking human placental DNA 1 μg/ml), Probemixture is denatured for 10 minutes in 70° C. water bath and incubatedfor 1 hour at 37° C., before placing on a prewarmed (37° C. ) slide,which is previously denatured in 70% formamide/2×SSC at 70° C., anddehydrated in ethanol series, chilled to 4° C.

Slides are incubated for 16 hours at 37° C. in a humidified chamber.Slides are ished in 50% formamide/2×SSC for 10 minutes at 41° C. and2×SSC for 7 minutes at 37° C. Hybridization probe is detected byincubation of the slides with FITC-Avidin (ONCOR, Gaithersberg, Md.),according to the manufacturer protocol. Chromosomes are counterstainedwith propridium iodine suspended in mounting medium. Slides arevisualized using a Leitz ORTHOPLAN 2-epifluorescence microscope and fivecomputer images are taken using Imagenetics Computer and Macintoshprinter.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, which is publicly available on line viacomputer. The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (Co-Inheritance of Physically Adjacent Genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Chromosome assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a CGRP-RCF gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA the is usedfor in situ chromosome mapping using well known techniques for thispurpose. Typically, in accordance with routine procedures for chromosomemapping, some trial and error may be necessary to identify a genomicprobe that gives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3' untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,because primers that span more than one exon in the genomic DNA couldcomplicate the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization ("FISH") of a cDNA clone to ametaphase chromosomal spread, as described above, can be used to providea precise chromosomal location in one step. This technique can be usedwith cDNA as short as 50 or 60. For a review of this technique, seeVerma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, PergamonPress, New York (1988).

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Polypeptide assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of CGRP-RCFprotein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of CGRP-RCF proteincompared to normal control tissue samples may be used to detect thepresence of diabetes, migrane, pain and inflammation, Parkinson'sdisease, acute heart failure, hypotension, urinary retention,osteoporosis, hypertension, angina pectoris, myocardial infarction,ulcers, asthma, allergies, psychosis, depression, vomiting, benignprostatic hypertrophy, Paget's disease, obesity, cancer, gigantism andthe like, for example. Assay techniques that can be used to determinelevels of a protein, such as an CGRP-RCF protein of the presentinvention, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to CGRP-RCF, preferably amonoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached a detectable reagent such as radioactive, fluorescent orenzymatic reagent, in this example horseradish peroxidase enzyme.

To carry out an ELISA a sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attach to any CGRP-RCF proteins attached tothe polystyrene dish. Unbound monoclonal antibody is ished out withbuffer. The reporter antibody linked to horseradish peroxidase is placedin the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to CGRP-RCF. Unattached reporter antibody isthen ished out. Reagents for peroxidase activity, including acolorimetric substrate are then added to the dish. Immobilizedperoxidase, linked to CGRP-RCF through the primary and secondaryantibodies, produces a colored reaction product. The amount of colordeveloped in a given time period indicates the amount of CGRP-RCFprotein present in the sample. Quantitative results typically areobtained by reference to a standard curve.

A competition assay may be employed wherein antibodies specific toCGRP-RCF attached to a solid support and labeled CGRP-RCF and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of CGRP-RCF in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, et al., Nature256:495-497 (1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72 (1983) and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985)).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, antibodies against CGRP-RCF may be employed toinhibit diabetes, migrane, pain and inflammation, Parkinson's disease,acute heart failure, hypotension, urinary retention, osteoporosis,hypertension, angina pectoris, myocardial infarction, ulcers, asthma,allergies, psychosis, depression, vomiting, benign prostatichypertrophy, Paget's disease, obesity, cancer, gigantism and the like.

CGRP-RCF binding molecules and assays

This invention also provides a method for identification of molecules,such as binding molecules, that bind CGRP-RCF/CGRP receptor. Genesencoding proteins that bind CGRP-RCF-CGRP receptor, such as bindingmolecules, can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting. Such methodsare described in many laboratory manuals such as, for instance, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. Tothis end polyadenylated RNA is prepared from a cell responsive toCGRP-RCF and CGRP receptor, a cDNA library is created from this RNA, thelibrary is divided into pools and the pools are transfected individuallyinto cells that are not responsive to α- or β-CGRP. The transfectedcells then are exposed to labeled α- or β-CGRP. (α- or β-CGRP can belabeled by a variety of well-known techniques including standard methodsof radio-iodination or inclusion of a recognition site for asite-specific protein kinase.) Following exposure, the cells are fixedand binding of α- or β-CGRP is determined. These procedures convenientlyare carried out on glass slides.

Pools are identified of cDNA that produced α- or β-CGRP binding cells.Sub-pools are prepared from these positives, transfected into host cellsand screened as described above. Using an iterative sub-pooling andre-screening process, one or more single clones that encode the putativebinding molecule, such as a receptor molecule, can be isolated.

Alternatively a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a receptor molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis("PAGE") and exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative binding molecule.

Polypeptides of the invention also can be used to assess CGRP-RCFbinding capacity of CGRP-RCF binding molecules in cells or in cell-freepreparations.

Agonists and antagonists--assays and molecules

The CGRP-RCF of the present invention may be employed in a process forscreening for compounds which activate (agonists) or inhibit activation(antagonists) of the CGRP receptor or (function of) CGRP-RCF or evenCGRP-RCF/CGRP receptor system.

In general, such screening procedures involve providing appropriatecells which express the CGRP-RCF/CGRP receptor on the surface thereof.Such cells include cells from mammals, yeast, drosophila or E. Coli. Inparticular, polynucleotides encoding the receptor system of the presentinvention are employed to transfect cells to thereby express theCGRP-RCF/CGRP receptor. The expressed polypeptides are then contactedwith a test compound to observe binding, stimulation or inhibition of afunctional response.

One such screening procedure involves the use of melanophores which aretransfected to express the CGRP-RCF/CGRP receptor. Such a screeningtechnique is described in PCT WO 92/01810 published Feb. 6, 1992.

Thus, for example, such assay may be employed for screening for acompound which inhibits activation of the CGRP-RCF/CGRP receptor bycontacting the melanophore cells which encode the polypeptides with boththe CGRP receptor ligand and a compound to be screened. Inhibition ofthe signal generated by the ligand indicates that a compound is apotential antagonist for the receptor system, i.e., inhibits activationof the CGRP-RCF/CGRP receptor.

The screen may be employed for determining a compound which activatesthe receptor system by contacting such cells with compounds to bescreened and determining whether such compound generates a signal, i.e.,activates the CGRP-RCF/CGRP receptor.

Other screening techniques include the use of cells which express theCGRP-RCF-CGRP receptor (for example, transfected CHO cells) in a systemwhich measures extracellular pH changes caused by CGRP-RCF/CGRP receptoractivation, for example, as described in Science, 246:181-296 (October1989). For example, compounds may be contacted with a cell whichexpresses the CGRP-RCF/CGRP receptor and a second messenger response,e.g. signal transduction or pH changes, may be measured to determinewhether the potential compound activates or inhibits the CGRP-RCF/CGRPreceptor.

Another such screening technique involves introducing RNAs encoding bothCGRP-RCF and CGRP receptor into Xenopus oocytes to transiently expressthe CGRP-RCF/CGRP receptor. The oocytes may then be contacted with theCGRP receptor ligand and a compound to be screened, followed bydetection of inhibition or activation of a calcium, proton, etc. signalin the case of screening for compounds which are thought to inhibitactivation of the receptor.

Another screening technique involves expressing the CGRP-RCF/CGRPreceptor in which the receptor system is linked to, for example, aphospholipase C or D or other proteins. As representative examples ofsuch cells, there may be mentioned endothelial cells, smooth musclecells, embryonic kidney cells, etc. The screening may be accomplished ashereinabove described by detecting activation of the receptor system orinhibition of activation of the receptor system from a second signal,such as for example phospholipase or other activated/expressed protein.

Another method involves screening for compounds which inhibit activationof the receptor system of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor system on the surface thereof. Such a method involvestransfecting a eukaryotic cell with DNAs encoding both CGRP-RCF and CGRPreceptor such that the cell expresses the receptor system on its surfaceand contacting the cell with a compound in the presence of a labeledform of a known ligand. The ligand can be labeled, e.g., byradioactivity. The amount of labeled ligand bound to the receptors ismeasured, e.g., by measuring radioactivity of the receptor system. Ifthe compound binds to the receptor system as determined by a reductionof labeled ligand which binds to the receptor system, the binding oflabeled ligand to the receptor system is inhibited.

Another method involves screening for CGRP-RCF/CGRP receptor inhibitorsby determining inhibition of CGRP-RCF/CGRP receptor-mediated cAMP and/oradenylate cyclase accumulation. Such a method involves transfecting aeukaryotic cell with the CGRP-RCF/CGRP receptor system to express thereceptor system on the cell surface. The cell is then exposed topotential antagonists in the presence of the receptor system. The amountof cAMP accumulation is then measured. If the potential antagonist bindsthe receptor system, and thus inhibits ligand binding, the levels ofCGRP-RCF/CGRP receptor mediated cAMP, or adenylate cyclase, activitywill be reduced.

The present invention also provides a method for determining whether aligand, not known to be capable of binding to a CGRP receptor orCGRP-RCF/CGRP receptor, can bind to such receptor or receptor systemwhich comprises contacting a mammalian cell which expressesCGRP-RCF/CGRP receptor with the CGRP receptor ligand under conditionspermitting binding of ligands to the CGRP-RCF/CGRP receptor system,detecting the presence of a ligand which binds to the receptor andthereby determining whether the ligand binds to the CGRP-RCF/CGRPreceptor. The systems hereinabove described for determining agonistsand/or antagonists may also be employed for determining ligands whichbind to the receptor system.

Examples of potential CGRP-RCF or CGRP-RCF/CGRP receptor antagonists arealso an antibody, or in some cases an oligonucleotide, which binds tothe CGRP-RCF or CGRP-RCF/CGRP receptor such that the activity of thepolypeptide is prevented.

Potential antagonists also include proteins which are closely related tothe ligand of the CGRP-RCF/CGRP receptor, i.e. a fragment of the ligand,which have lost biological function and when binding to the receptorsystem, elicit no response.

A potential antagonist also includes an antisense construct preparedthrough the use of antisense technology. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5' coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix--see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of CGRP-RCF. The antisense RNA oligonucleotide hybridizesto the mRNA in vivo and blocks translation of the mRNA molecule into theCGRP-RCF (antisense--Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the CGRP-RCF.

Another potential antagonist is a small organic molecule which binds tothe CGRP-RCF or CGRP-RCF/CGRP receptor, making it inaccessible toligands such that its normal biological activity is prevented.

Potential antagonists also include a soluble form of a CGRP-RCF, e.g. afragment, which binds to the CGRP receptor ligand and prevents theligand from interacting with membrane bound CGRP-RCF/CGRP receptor.

CGRP-RCF are responsible for many biological functions, including manypathologies. Accordingly, it is desirous to find compounds and drugswhich stimulate the CGRP-RCF or CGRP-RCF/CGRP receptor on the one handand which can inhibit the function of a CGRP-RCF or CGRP-RCF/CGRPreceptor on the other hand.

In general, agonists for CGRP-RCF or CGRP-RCF/CGRP receptor are employedfor prophylaxis of or for treating Parkinson's disease, acute heartfailure, hypotension, urinary retention.

Antagonists for CGRP-RCF or CGRP-RCF/CGRP receptor may be employed forprophylaxis of or for treating hypertension, angina pectoris, myocardialinfarction, ulcers, asthma, allergies, psychoses, depression, migrane,vomiting, and benign prostatic hyertrophy.

The agonists may also be employed to treat osteoporosis since CGRPinhibits osteoclast-mediated bone resorption and stimulatesosteogenesis. In this same manner hypercalcemia may be treated.Similarly, the agonists may be employed to treat Paget's Disease.

The agonists may also be employed to stimulate angiogenesis and promotewound healing via the stimulatory effect of CGRP on endothelial cellproliferation.

The agonists may also be employed to treat obesity since CGRP controlsfeeding behavior by decreasing appetite and intestinal motility.

The agonists may also be employed to stimulate nerve regeneration sinceCGRP is a trophic agent in the CNS.

The agonists may also be employed to enhance the immune response throughincreasing vascular permeability.

Agonists may also be employed to inhibit superoxide production.Superoxide production is known in the art to cause cellular damage andlead possibly to diseases such as cancer.

The antagonists may also be employed to inhibit CNS pain transmission,to treat chronic inflammation caused by long-lived vasodilation,arthritis, maturity onset diabetes, cardiovascular disorders and totreat migraine headaches.

The antagonists may also be employed to prevent carcinoid tumor of thelung, since an elevated level of CGRP has been found in the lung duringthis disease.

This invention additionally provides a method of treating an abnormalcondition related to an excess of CGRP-RCF activity which comprisesadministering to a subject the inhibitor compounds (antagonists) ashereinabove described along with a pharmaceutically acceptable carrierin an amount effective to inhibit activation by blocking binding ofligands to the CGRP-RCF/CGRP receptor, or by inhibiting a second signal,and thereby alleviating the abnormal conditions.

The invention also provides a method of treating abnormal conditionsrelated to an under-expression of CGRP-RCF activity which comprisesadministering to a subject a therapeutically effective amount of acompound which activates the receptor system of the present invention(agonists) as described above in combination with a pharmaceuticallyacceptable carrier, to thereby alleviate the abnormal conditions.

Compositions and Kits

The soluble form of the CGRP-RCF, and compounds which activate orinhibit such polypeptide receptor system, may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide or compound, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above. Thus, the polypeptides of thepresent invention may be employed in combination with a non-sterile orsterile carrier or carriers for use with cells, tissues or organisms,such as a pharmaceutical carrier suitable for administration to asubject. Such compositions comprise, for instance, a media additive or atherapeutically effective amount of a polypeptide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration.

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Gene therapy

The CGRP-RCF polynucleotides, polypeptides, agonists and antagoniststhat are polypeptides may be employed in accordance with the presentinvention by expression of such polypeptides in vivo, in treatmentmodalities often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein abovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, arian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors well include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retrovirai LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller et al., Biotechniques7:980-990 (1989), or any other promoter (e.g., cellular promoters suchas eukaryotic cellular promoters including, but not limited to, thehistone, RNA polymerase III, and B-actin promoters). Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegaiovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, A., Human Gene Therapy 1:5-14(1990). The vector may be transduced into the packaging cells throughany means known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO4 precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples are carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), herein referred to as "Sambrook."

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow is carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis ("PAGE") in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

Unless described otherwise, ligations are accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase ("ligase") per 0.5 μg of DNA.

Example 1

Expression and purification of human CGRP-RCF using bacteria

The DNA sequence encoding human CGRP-RCF in the deposited polynucleotideis digested with specific endonuclease restriction enzyme EcoRI and XhoIto release a fragment encoding the open reading frame of CGRP-RCF andthe fragment is purified and ligated into the bacterial expressionvector, which is digested with the appropriate enzymes.

The restrictions sites are convenient to restriction enzyme sites in thebacterial expression vectors pQE-9 which are used for bacterialexpression in these examples. (Qiagen, Inc. Chatsworth, Calif. pQE-9encodes ampicillin antibiotic resistance ("Ampr") and contains abacterial origin of replication ("ori"), an IPTG inducible promoter, aribosome binding site ("RBS"), a 6-His tag and restriction enzyme sites.

The human CGRP-RCF DNA and the vector pQE-9 both are digested with EcoRIand XhoI and the digested DNAs then are ligated together. Insertion ofthe CGRP-RCF DNA into the EcoRI/XhoI restricted vector placed theCGRP-RCF coding region downstream of and operably linked to the vector'sIPTG-inducible promoter and in-frame with an initiating AUGappropriately positioned for translation of CGRP-RCF.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance ("Kan^(r) "),is used in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressingCGRP-RCF, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin. Plasmid DNA is isolated from resistantcolonies and the identity of the cloned DNA is confirmed by restrictionanalysis.

Clones containing the desired constructs are grown overnight ("O/N") inliquid culture in LB media supplemented with both ampicillin (100 ug/ml)and kanamycin (25 ug/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm ("OD⁶⁰⁰ ") of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside ("IPTG") is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate buffered saline("PBS"), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2× PBS at a concentration of 95micrograms per mL.

Example 2

Cloning and expression of human CGRP-RCF in a baculovirus expressionsystem

The cDNA sequence encoding the full length human CGRP-RCF protein, inthe deposited clone is digested with EcoRI/XhoI to release the CGRP-RCFfragment. The fragment is then inserted into expression vector, whichwas also digested with the same restriction enzymes.

The vector pRG1 is used to express the CGRP-RCF protein in thebaculovirus expression system, using standard methods, such as thosedescribed in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORSAND INSECT CELL CULTURE PROCEDURES, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987). This expression vector contains thestrong polyhedrin promoter of the Autographa californica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sites.The signal peptide of AcMNPV gp67, including the N-terminal methionine,is located just upstream of a BamH1 site. The polyadenylation site ofthe simian virus 40 ("SV40") is used for efficient polyadenylation. Foran easy selection of recombinant virus the beta-galactosidase gene fromE.coli is inserted in the same orientation as the polyhedrin promoterand is followed by the polyadenylation signal of the polyhedrin gene.The polyhedrin sequences are flanked at both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pRG1, such aspac373, pVL941 and pAcIM1 provided, as those of skill in the an willreadily appreciate, that construction provides appropriately locatedsignals for transcription, translation, trafficking and the like, suchas an in-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170:31-39, among others.

The plasmid is digested with the restriction enzymes EcoRI and XhoI andthen is dephosphorylated using calf intestinal phosphatase, usingroutine procedures known in the art. The DNA is then isolated from a 1%agarose gel using a commercially available kit ("Geneclean" BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein "V2".

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E.coli HB101 cells are transformed with ligation mixand spread on culture plates. Bacteria are identified that contain theplasmid with the human CGRP-RCF gene by digesting DNA from individualcolonies using EcoRI and XhoI and then analyzing the digestion productby gel electrophoresis. The sequence of the cloned fragment is confirmedby DNA sequencing. This plasmid is designated herein pBacCGRP-RCF.

5 μg of the plasmid pBacCGRP-RCF is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA ("BaculoGold™baculovirus DNA", Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). llag of BaculoGold™ virus DNA and 5 μg of theplasmid pBacCGRP-RCF are mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a "plaqueassay" of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted CGRP-RCF is identified by DNA analysisincluding restriction mapping and sequencing. This is designated hereinas V-CGRP-RCF.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-CGRP-RCF at a multiplicity of infection ("MOI") of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of35S-methionine and 5 μCi 35S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3

Gene therapeutic expression of human CGRP-RCF

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted--the chunks of tissue remain fixed to the bottom of theflask--and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes forcloning a fragment to be expressed. The digested vector is treated withcalf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

CGRP-RCF cDNA capable of expressing active CGRP-RCF, is isolated. Theends of the fragment are modified, if necessary, for cloning into thevector. For instance, 5' overhanging may be treated with DNA polymeraseto create blunt ends. 3' overhanging ends may be removed using S1nuclease. Linkers may be ligated to blunt ends with T4 DNA ligase.

Equal quantities of the Moloney murine leukemia virus linear backboneand the CGRP-RCF fragment are mixed together and joined using T4 DNAligase. The ligation mixture is used to transform E. coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

Packaging cells are grown in tissue culture to confluent density inDulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the CGRP-RCF gene isintroduced into the packaging cells by standard techniques. Infectiousviral particles containing the CGRP-RCF gene are collected from thepackaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriateincubation period media is harvested from the plates of confluentproducer cells. The media, containing the infectious viral particles, isfiltered through a Millipore filter to remove detached producer cells.The filtered media then is used to infect fibroblast cells. Media isremoved from a sub-confluent plate of fibroblasts and quickly replacedwith the filtered media. Polybrene (Aldrich) may be included in themedia to facilitate transduction. After appropriate incubation, themedia is removed and replaced with fresh media. If the liter of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his, toselect out transduced cells for expansion.

Engineered fibroblasts then may be injected into rats, either alone orafter having been grown to confluence on microcarrier beads, such ascytodex 3 beads. The injected fibroblasts produce CGRP-RCF product, andthe biological actions of the protein are conveyed to the host.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

Example 4

In Vitro Transcription, Microinjection into Xenopus laevis oocytes andElectrophysiology

Capped RNA transcripts were synthesized from linearized pCGRP-RCF orpCGRP-type-I-receptor DNA using T7 RNA polymerase (Stratagene). Formicroinjection, X. laevis oocytes were prepared as previously described(Elshourbagy et al., J. Biol. Chem., 268:3873-3879 (1993)). Stage V-VIoocytes were selected, and the follicular membranes were manuallyremoved. For each experimental group, 6 defolliculated oocytes werecoinjected with 50 nl of water containing 20 ng of CGRP-RCF and CGRPtype I receptor complementary RNA (cRNA) (Drummond injection apparatus).The injected oocytes were maintained in modified Barth's medium at 18°C. for 48 hrs to allow for protein synthesis complex to the cellsurface. Electrophysiology was performed using the voltage clamptechnique using an oocyte voltage clamp apparatus (Warner Instruments).Oocytes were clamped at -60 mV, and exposed to 10⁻⁷ M human alpha-CGRPor human Calcitonin (Bachere) and the Ca⁺² activated channel activitywas recorded in Barth's medium at room temperature.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1450 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCACGAGCAGCTGTGAAGTGTGAGGTTCTTTGTCTGCTGGCAGCTAGGGGCGACGAGGC60                GGGACGTCATGGAAGTGAAGGATGCCAATTCTGCGCTTCTCAGTAACTACGAGGTATTTC120               AGTTACTAACTGATCTGAAAGAGCAGCGTAAAGAAAGTGGAAAGAATAAACACAGCTCTG180               GGCAACAGAACTTGAACACTATCACCTATGAAACGTTAAAATACATATCAAAAACACCAT240               GCAGGCACCAGAGTCCTGAAATTGTCAGAGAATTTCTCACAGCATTGAAAAGCCACAAGT300               TGACCAAAGCTGAGAAGCTCCAGCTGCTGAACCACCGGCCTGTGACTGCTGTGGAGATCC360               AGCTGATGGTGGAAGAGAGTGAAGAGCGGCTCACGGAGGAGCAGATTGAAGCTCTTCTCC420               ACACCGTCACCAGCATTCTGCCTGCAGAGCCAGAGGCTGAGCAGAAGAAGAATACAAACA480               GCAATGTGGCAATGGACGAAGAGGACCCAGCATAGAAGAGCACAGCTGGCCCCGGCGTTT540               CATGAAGTCAGAAGGCCTGGCAGCCATTTCCTGGACGTTGAGAGGATTGTTTATTTGATT600               TTTATCCTCATCCCAGCAGGCCTGGCTTTGTGGTTAGTTGGGTACATCACAAAAATAAGT660               TAAAAAGAAATATTTGTGCCTTGGGGAGAAGAAACATGGTGAAAACAGGCTGAGGTTGTC720               AGGGCAGAGAGCTGAAGGTGGGGACAGTGACCGCGGACCCCTCTGCGCTTGAAAGATTTC780               CTCCACGGCCTTTGCCCCAGTTGTGGGGAGGTCTCTGTGCACAGCGGGGAAAATGCTTGT840               GTCGCCTTTGGTGGGCCATGTCCTAATTAGTTTCATCTGCTTCCCTGGGAACTTACTAAG900               GGGCCCAGAGCACTGTTGGAAGTCTGGTTAGAGTCCCCAGAGAGTTACTCTAAGTTAAAA960               TGAGCCACTGACCTTGGCTCACCTTAGAGGAATTTCCTCGAGAACAACAGAGATAAGAAA1020              AGAACCGGCCTGGCCAATCCTTCAACAGCTCTAGAGCCCCTTTTCTCTGCTGGCAGGGGC1080              TTTGTTTACCAGCTCACTGTTTAGGCTAAATGTTAGGGACCAGATCACTGCAGTTGAAAA1140              CAGCATCCAGGCTTAGTGACAGTGGCAGCAGAAACAGTGTTGGCTGCCTTTCTGACCACC1200              CCACTTTCCTGCCCTGAGACAGCAGCCCAGGGCAGGTGCTTCATATTCAGACCAGGTAAG1260              CCTCATTTGCACAACAGTCAAATTGTTTGTTCCTTTAAAAAGGACACAATTAGCCTGGCA1320              CGGTGACTCATGCTTGTAATCCCAGCACTTTGGGAGGGCAAGGCAGGCGGATCACCTGAG1380              GTCAGGAGTTTGAGACCAGCCTCACCAACATGGAAAAACCCCATCTCTATTAAAAAAAAA1440              AAAAAAAAAA1450                                                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 148 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluValLysAspAlaAsnSerAlaLeuLeuSerAsnTyrGluVal                              151015                                                                        PheGlnLeuLeuThrAspLeuLysGluGlnArgLysGluSerGlyLys                              202530                                                                        AsnLysHisSerSerGlyGlnGlnAsnLeuAsnThrIleThrTyrGlu                              354045                                                                        ThrLeuLysTyrIleSerLysThrProCysArgHisGlnSerProGlu                              505560                                                                        IleValArgGluPheLeuThrAlaLeuLysSerHisLysLeuThrLys                              65707580                                                                      AlaGluLysLeuGlnLeuLeuAsnHisArgProValThrAlaValGlu                              859095                                                                        IleGlnLeuMetValGluGluSerGluGluArgLeuThrGluGluGln                              100105110                                                                     IleGluAlaLeuLeuHisThrValThrSerIleLeuProAlaGluPro                              115120125                                                                     GluAlaGluGlnLysLysAsnThrAsnSerAsnValAlaMetAspGlu                              130135140                                                                     GluAspProAla                                                                  145                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide encoding an amino acid sequence having at least 95%identity to the calcitonin gene-related peptide-receptor componentfactor (CGRP-RCF) of SEQ ID NO:2, wherein said amino acid sequenceconfers CGRP responsiveness in Xenopus oocytes expressing the CGRPreceptor; or a polynucleotide which is fully complementary to saidisolated polynucleotide.
 2. A polynucleotide of claim 1 which by virtueof the redundancy of the genetic code encodes the amino acid sequence ofSEQ ID NO:2; or a polynucleotide which is fully complementary to saidpolynucleotide.
 3. The polynucleotide of claim 1 wherein thepolynucleotide is RNA.
 4. The polynucleotide of claim 1 wherein thepolynucleotide is DNA.
 5. The polynucleotide of claim 4 as set forth inSEQ ID NO:1.
 6. The polynucleotide of claim 4 comprising nucleotide 69to 512 as set forth in SEQ ID NO:1.
 7. The polynucleotide of claim 4which encodes a polypeptide comprising the amino acids set forth in SEQID NO:2.
 8. A vector comprising the DNA of claim
 4. 9. A host cellcomprising the vector of claim
 8. 10. A process for producing a CGRP-RCFprotein comprising: expressing from the host cell of claim 9 a CGRP RCFprotein encoded by said DNA.
 11. A process for producing a cell whichexpresses a CGRP-RCF protein comprising transforming or transfecting acell with the vector of claim 8 such that the cell expresses a CGRP-RCFprotein.
 12. An isolated polynucleotide comprising a nucleotide sequencehaving at least 95% identity to the human cDNA CGRP-RCF encodingsequence contained in ATCC Deposit No. 98105, wherein said nucleotidesequence encodes an amino acid sequence that confers CGRP responsivenessin Xenopus oocytes expressing the CGRP receptor; or a polynucleotidewhich is fully complimentary to said isolated polynucleotide.
 13. Apolynucleotide of claim 12 which by virtue of the redundancy of thegenetic code encodes the same mature CGRP-RCF protein expressed by thehuman cDNA contained in ATCC No 98105; and a polynucleotide fullycomplementary to said polynucleotide.